The Gram-positive bacterium Listeria monocytogenes (Lm) is a facultative intracellular pathogen that relies on the regulated secretion and activity ofa variety of gene products to promote life within diverse host environments. Our lab recently identified a protein known as PrsA2, a secreted chaperone that is dispensable for bacterial growth in broth culture but essential for Lm virulence. PrsA2 is a peptidyl-prolyl cis/trans isomerase that contributes to the stability and activity of a number of Lm secreted virulence factors that are required for bacterial invasion, replication, and cell-to-cell spread within the infected host. We hypothesize that during host infection PrsA2 regulates the proper folding, stabilization, and activity of secreted proteins that contribute to bacterial virulence and viabiliy within the host cytosol. Despite the pivotal importance of secreted proteins in bacterial pathogenesis, little is known about the mechanisms that regulate protein activity following membrane translocation in Gram-positive bacteria. PrsA homologs in other species appear to be required for the folding and stability of secreted protein factors at the membrane-cell wall interface. Lm has two prsA alleles (prsA1 and prsA2) and we have identified the first genetic link between these two alleles, a novel two-component regulatory system (Lmo1507-1508) that regulates the expression of both prsA1 and prsA2. PrsA1 shares 75% amino acid similarity and 58% identity with PrsA2, and we have solved the crystal structure for PrsA1, a novel structure that we have used to model PrsA2 in order to identify structural changes between the two proteins that may determine functional differences. My research objectives are to mechanistically define the role of PrsA2 and PrsA1 in Lm physiology and pathogenesis and to define the molecular mechanism that links prsA1 and prsA2. Data obtained from these experiments will provide new mechanistic insight into how a critical secretion chaperone carries out its activities and is responsive to environmental signals.